Telomeres pose a challenge to the replication machinery due to the repetitive nature of the DNA and its unusual terminal structure. As a result, telomere replication requires the action of ancillary factors in addition to the conventional replication machinery. Mechanistic understanding of how the actions of these factors are coordinated is still lacking and additional factors are being discovered. During the previous grant period the applicant identified a new telomere replication factor, the human CST (CTC1-STN1-TEN1) complex. CST has independent functions during telomere duplex replication and during fill-in synthesis of the telomeric C- strand after G-strand elongation by telomerase. CST also facilitates new origin firing during genome-wide replication restart after replication fork stalling. Moreover, mutations in the CTC1 subunit of CST cause dyskeratosis congenita and the neurological disorder Coats plus. CTC1 and STN1 correspond to AAF, a previously identified polymerase (pol ) stimulatory factor. The objective of this proposal is to determine the mechanism of CST action during telomere replication and hence to understand how deficiencies in CTC1 lead to human disease. The central hypotheses is that CST enables pol to (re-)initiate DNA synthesis after replication fork stalling in the telomere duplex and during maturation of the telomeric 3' overhang. We propose that CST functions in conjunction with shelterin/TPP1 and telomeric DNA to recruit or load pol for C-strand fill-in at the 3' overhang and that CST facilitates new origin firing or fork restart within the telomere duplex. Aim 1 will define CST structure and function by analysis of disease-related and DNA-binding mutations. Experiments will examine the effects of these mutations on telomere replication and CST biochemistry. Aim 2 will determine how CST directs pol activity at the telomeric 3' overhang. In vitro studies will examine how CST activity is modified by telomere components while in vivo studies will examine the importance of CST, pol and TPP1 interactions for pol -mediated C-strand synthesis. Aim 3 will determine how CST functions in telomere duplex replication. The replication barriers overcome by CST action will be defined and the pathway for replication rescue will be identified. Outcome: Collectively the three aims will provide essential insight into the mechanism of action of a novel telomere replication factor and will answer fundamental questions about how this factor prevents telomere loss and human disease. The work will reveal how CTC1 architecture impacts CST function and will uncover why CTC1 patient mutations cause Coats plus or dyskeratosis congenita. It will lead to a new understanding of how CST enables the cell to overcome replication barriers within the telomere to prevent telomere loss and how CST permits pol to complete replication of the chromosome terminus. The significance is that the work will provide key information about aspects of telomere replication that directly impact human health. This information is needed for better diagnosis and treatment of telomere-related disease.